Rhodamine compounds and their use as fluorescent labels

ABSTRACT

The present invention relates to new rhodamine compounds and their use as fluorescent labels. The compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/774,898, filed on Mar. 8, 2013, which is hereby incorporated byreference in its entirety.

The present invention relates to new rhodamine compounds and their useas fluorescent labels. The compounds may be used as fluorescent labels,particularly for nucleotide labelling in nucleic acid sequencingapplications.

BACKGROUND

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of thesepublications and documents is incorporated by reference herein.

Non-radioactive detection of nucleic acids utilizing fluorescent labelsis an important technology in molecular biology. Many proceduresemployed in recombinant DNA technology previously relied heavily on theuse of nucleotides or polynucleotides radioactively labelled with, forexample ³²P. Radioactive compounds permit sensitive detection of nucleicacids and other molecules of interest. However, there are seriouslimitations in the use of radioactive isotopes such as their expense,limited shelf life and more importantly safety considerations.Eliminating the need for radioactive labels enhances safety whilstreducing the environmental impact and costs associated with, forexample, reagent disposal. Methods amenable to non-radioactivefluorescent detection include by way of non-limiting example, automatedDNA sequencing, hybridization methods, real-time detection ofpolymerase-chain-reaction products and immunoassays.

For many applications it is desirable to employ multiple spectrallydistinguishable fluorescent labels in order to achieve independentdetection of a plurality of spatially overlapping analytes. In suchmultiplex methods the number of reaction vessels may be reducedsimplifying experimental protocols and facilitating the production ofapplication-specific reagent kits. In multi-colour automated DNAsequencing for example, multiplex fluorescent detection allows for theanalysis of multiple nucleotide bases in a single electrophoresis lanethereby increasing throughput over single-colour methods and reducinguncertainties associated with inter-lane electrophoretic mobilityvariations.

However, multiplex fluorescent detection can be problematic and thereare a number of important factors which constrain selection offluorescent labels. First, it is difficult to find dye compounds whoseemission spectra are suitably spectrally resolved. In addition whenseveral fluorescent dyes are used together, simultaneous excitation maybe difficult because the absorption bands of the dyes are usually widelyseparated. Many excitation methods use high power lasers and thereforethe dye must have sufficient photo-stability to withstand such laserexcitation. A final consideration of particular importance in molecularbiology methods is that the fluorescent dyes must be compatible with thereagent chemistries used such as for example DNA synthesis solvents andreagents, buffers, polymerase enzymes and ligase enzymes.

As sequencing technology advances a need has developed for furtherfluorescent dye compounds, their nucleic acid conjugates and dye setswhich satisfy all of the above constraints and which are amenableparticularly to high throughput molecular methods such as solid phasesequencing and the like.

Application WO2007135368 describes a class of rhodamine compoundssuitable for use as fluorescent labels. The compounds described thereinare suitable for use in solid phase nucleic acid sequencing protocols.Advances in the technology and throughput of solid phase nucleic acidsequencing have led to further developments and improvements to themolecular design of fluorescent labels, particularly in the context ofthe interaction between the fluorescent reagents and particular nucleicacid sequences.

Fluorescent dye molecules with improved fluorescence properties (such asfluorescence intensity, position of fluorescence maximum and shape offluorescence band) can improve the speed and accuracy of nucleic acidsequencing. Fluorescence signal intensity is especially important whenmeasurements are made in water based biological buffers and/or at highertemperature as fluorescence of most dyes is significantly lower at suchconditions. Moreover, the nature of the base to which a dye is attachedalso affects the fluorescence maximum, fluorescence intensity and otherspectral dye properties. The sequence specific interactions between thefluorescent dye and the nucleobase can be tailored by specific design ofthe fluorescent dyes. Optimisation of the structure of the fluorescentdyes can improve their fluorescent properties and also improve theefficiency of nucleotide incorporation, reduce the level of sequencingerrors and decrease the usage of reagents in, and therefore the costsof, nucleic acid sequencing.

Described herein are improved rhodamine constructs and their use asbio-molecule labels, particularly as labels for nucleotides used innucleic acid sequencing. The improvements can be seen in the higherfluorescence intensities of such dyes when prepared as bio-moleculeconjugates and in the length and quality of sequencing read obtainableusing the new fluorescent constructs.

SUMMARY

According to a first aspect the invention provides rhodamine dyecompounds of the formula (I) and mesomers thereof:

Mesomers of the invention may include formula's represented by 1a, 1b orcyclic form 1c:

In formula's I, Ia, Ib or Ic,

M^(+/−) is a common counter ion,

k is an integer of from 0 to 6,

q is an integer of from 1 to 6,

Y═O, NR₁₁,

R₁ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,

R₂ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group, or R₂ together with R₁ or R₅ is acarbon or heterosubstituted chain forming a ring,

R₃ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group or R₃ together with R₄ or R₆ is acarbon or heterosubstituted chain forming a ring,

R₄ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,

R₅ and R₆ are H, alkyl or substituted alkyl group, halogen, hydroxy- oralkoxy group,

R₈ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₁ is a carbon or heterosubstituted carbon chainforming a ring,

R₉ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₄ is a carbon or heterosubstituted carbon chainforming a ring,

R₇ is OR₁₁ or NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or asubstituted alkyl,

R₁₃ is OR₁₄ or NR₁₄R₁₅ where R₁₄ and R₁₅ are independently H, alkyl or asubstituted alkyl; aryl or a substituted aryl, and

R₁₆ and R₁₇ are independently H or an alkyl, aryl or substituted alkylor substituted aryl group.

In another embodiment the compounds of the present invention can beconjugated with a variety of substrate moieties such as, for example,nucleosides, nucleotides, polynucleotides, polypeptides, carbohydrates,ligands, particles and surfaces.

According to a further aspect of the invention therefore, there areprovided dye compounds comprising linker groups to enable, for example,covalent attachment to such substrate moieties.

According to a further aspect the invention provides a nucleoside ornucleotide compound defined by the formula: N-L-Dye, wherein N is anucleotide, L is an optional linker moiety and Dye is a fluorescentcompound according to the present invention.

In a further aspect the invention includes methods of sequencing usingthe dye compounds of the present invention.

According to a further aspect the invention also provides kitscomprising dye compounds of the invention (free or in conjugate form)which may be used in various immunological assays, oligonucleotide andnucleic acid labelling and for DNA sequencing by synthesis. In yetanother aspect the invention provides kits comprising dye ‘sets’particularly suited to cycles of sequencing by synthesis on an automatedinstrument platform.

A further aspect of the invention is the chemical preparation ofcompounds of the invention.

DESCRIPTION OF FIGURES

FIG. 1 shows the lower temperature decrease of the dyes as describedherein compared to prior art dyes. Normalized fluorescence intensitiesof 1.10⁻⁶ M solutions of dyes (I-1) and (I-3) were compared withcommercially available dye Atto532 for the same spectral region atdifferent temperature. The intensity of the dyes at 20, 40 and 60° C.were measured. FIG. 1 shows the relative intensity of the dyes at eachtemperature. The commercial dye Atto532 shows a greater loss offluorescence intensity at higher temperatures relative to the I-1 andI-3 dyes. FIG. 1 demonstrates that the fluorescence of the new dyes inwater based solutions is less variable with the temperature.

FIG. 2 demonstrates that fluorescence of the nucleobase conjugates basedon these new dyes in water based solutions is higher than commerciallyavailable dyes when excited by 532 nm light. Normalised Fluorescencespectra of 1.10⁻⁶ M solutions of dye-nucleobase conjugates (I-13)-T and(I-11)-T were compared with structural analogue when pppT is conjugatedwith commercially available dye Atto532. The 1-3 dye is brighter thanthe atto 532 dye at the same nucleotide concentration. The I-1 dye isred shifted compared to the atto 532 dye.

FIG. 3 demonstrates that fluorescence of new dyes in water basedsolutions is less dependable on temperature. Normalized fluorescenceintensities 1.10⁻⁶ M solutions of dyes when conjugated with nucleobase,T-(I-11) and T-(I-13) as compared with commercially available dyeAtto532 conjugated with the same T-nucleobase. Both the I-1 and I-3 dyesshow a higher fluorescence intensity at elevated temperatures comparedto atto-532.

FIG. 4 demonstrates better distinguishing of fluorescence signals when anucleobase been labelled with the new dye in according with theinvention (I-3) (lane 6) as compared with standard fluorophore set whenthe same nucleobase been conjugated with commercially available dyeAtto532 (control 1). FIG. 4 shows a plot of red intensity vs greenintensity in an Illumina 4 colour sequencing run. The higher distancebetween the groups of dyes signals lowers the chances of a miss-call,and therefore increases the accuracy of sequencing. The increase inbrightness of the I-3 dye compared to the commercial dye means thesequencing data is improved.

DETAILED DESCRIPTION

The invention relates to novel rhodamine dye compounds particularlysuitable for methods of fluorescence detection and sequencing bysynthesis.

According to a first aspect the invention provides rhodamine dyecompounds of the formula (I):

whereinM^(+/−) is a common counter ion,k is an integer of from 0 to 6,q is an integer of from 1 to 6,R₁ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,R₂ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group, or R₂ together with R₁ or R₅ is acarbon or hetero-substituted chain forming a ring,R₃ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group or R₃ together with R₄ or R₆ is acarbon or hetero-substituted chain forming a ring,R₄ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,R₅ and R₆ are H, alkyl or substituted alkyl group, halogen, hydroxy- oralkoxy group,R₈ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₁ is a carbon or hetero-substituted carbon chainforming a ring,R₉ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₄ is a carbon or hetero-substituted carbon chainforming a ring,R₇ is OR₁₁ or NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or asubstituted alkyl,R₁₃ is OR₁₄ or NR₁₄R₁₅ where R₁₄ and R₁₅ are independently H, alkyl or asubstituted alkyl; aryl or a substituted aryl, andR₁₆ and R₁₇ are independently H or an alkyl, aryl or substituted alkylor substituted aryl group.

R₁₆ and R₁₇ can be independently H or an alkyl, aryl or substitutedalkyl or substituted aryl group. The alkyl group can be substituted withSO₃ ⁻. Where R₁₆ or R₁₇ is an alkyl substituted with SO₃ ⁻, the SO₃ ⁻group may be coordinated with a counter ion, for example metal ions orammonium ions. R₁₆ and R₁₇ can be H. Compounds of the inventiontherefore include compounds of formula (II) and mesomers thereof:

wherein M^(+/−) is a common counter ion,k is an integer of from 0 to 6,q is an integer of from 1 to 6,R₁ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,R₂ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group, or R₂ together with R₁ or R₅ is acarbon or hetero-substituted chain forming a ring,R₃ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group or R₃ together with R₄ or R₆ is acarbon or hetero-substituted chain forming a ring,R₄ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,R₅ and R₆ are H, alkyl or substituted alkyl group, halogen, hydroxy- oralkoxy group,R₈ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₁ is a carbon or heterosubstituted carbon chainforming a ring,R₉ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₄ is a carbon or heterosubstituted carbon chainforming a ring,R₇ is OR₁₁ or NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or asubstituted alkyl, andR₁₃ is OR₁₄ or NR₁₄R₁₅ where R₁₄ and R₁₅ are independently H, alkyl or asubstituted alkyl; aryl or a substituted aryl.

Where R₁₆ and/or R₁₇ is unsubstituted alkyl —(CH₂)_(n)H or —(CH₂)_(m)H,n and m may be 1-6. Compounds of the invention therefore includecompounds of formula (IIa):

wherein M⁻ is a common counter ion,k, n, and m are independently integers of from 0 to 6,q is an integer of from 1 to 6,R₁ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,R₂ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group, or R₂ together with R₁ or R₅ is acarbon or heterosubstituted chain forming a ring,R₃ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group or R₃ together with R₄ or R₆ is acarbon or heterosubstituted chain forming a ring,R₄ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,R₅ and R₆ are H, alkyl or substituted alkyl group, halogen, hydroxy- oralkoxy group,R₈ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₁ is a carbon or heterosubstituted carbon chainforming a ring,R₉ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₄ is a carbon or heterosubstituted carbon chainforming a ring,R₇ is OR₁₁ or NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or asubstituted alkyl, andR₁₃ is OR₁₄ or NR₁₄R₁₅ where R₁₄ and R₁₅ are independently H, alkyl or asubstituted alkyl; aryl or a substituted aryl.

n and m may the same or different. n may be 1, 2, 3, 4, 5 or 6. m may be1, 2, 3, 4, 5 or 6. The —(CH₂)n—H or —(CH₂)m—H may be C1-6 alkyl groups,for example methyl, ethyl or propyl, and may be optionally substituted.

q may be 1, 2, 3, 4, 5 or 6. In addition to the (CH₂)_(q) linker, thelinker may contain substituents at any carbon atoms or additionalheteroatoms. For example the linker may contain additional oxygen atomsin the form of ethylene glycol type spacers —(CH₂CH₂O)_(n)—. The linkeris present to attach the biomolecule through COR₁₃ residue in form of anacid, ester or amide group to the rest of the construct responsible forfluorescence and to separate the biomolecule from the dye molecule.

R₁ may be H or an alkyl or substituted alkyl group. R₁ can be chosensuch that R₁ may not be H when n is zero. R₁ can be methyl or ethyl. R₁can be linked to R₂ and/or R₈ to form a ring structure. The ring may bea 5 or 6 membered ring. The ring may be an all carbon ring, or maycontain additional heteroatoms.

R₂ may be H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group. Optionally, R₂ together with R₁or R₅ is a carbon or heterosubstituted chain forming a ring.

R₃ may be H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy or alkoxy group. Optionally

R₃ together with R₄ or R₆ is a carbon or hetero-substituted chainforming a ring.

R₄ may be H or an alkyl or substituted alkyl group. R₄ is not H when mis zero. R₄ can be methyl or ethyl. R₄ can be linked to R₃ and/or R₉ toform a ring structure. The ring may be a 5 or 6 membered ring. The ringmay be an all carbon ring, or may contain additional heteroatoms.

R₅ and R₆ may be H, alkyl or substituted alkyl group, halogen, hydroxyor alkoxy group. Optionally R₅ may be linked to R₂, and R₆ may be linkedto R₃.

R₈ may be H, halogen, hydroxy or alkoxy group, alkyl or substitutedalkyl group. Optionally R₈ may be linked together with R₁ to form acarbon or heterosubstituted carbon chain forming a ring.

R₉ may be H, halogen, hydroxy or alkoxy group, alkyl or substitutedalkyl group. Optionally R₉ may be linked together with R₄ to form acarbon or heterosubstituted carbon chain forming a ring.

R₇ together with the C═O forms an acid, ester or amide group. Inparticular R₇ may be OR₁₁ or NR₁₁R₁₂ where R₁₁ and R₁₂ are independentlyH, alkyl or a substituted alkyl, aryl or a substituted aryl. When R₁₁and R₁₂ are H R₇ may be OH or NH₂. R₇ may be an alkoxy group or aprimary or secondary amine group with one or two alkyl and/or arylgroups. The ester or amide COR₇ may be further optionally substituted.

R₁₃ together with the C═O is forms an acid, ester or amide group. Inparticular R₁₃ may be OR₁₄ or NR₁₄R₁₅ where R₁₄ and R₁₅ areindependently H, alkyl or a substituted alkyl, aryl or substituted aryl.R₁₃ may be OH or NH₂. R₁₃ may be an alkoxy or a primary or secondaryamine with one or two alkyl/aryl groups. The ester or amide may befurther optionally substituted. R₁₃ may be NR₁₄R₁₅ where R₁₄ is H oralkyl and R₁₅ is alkyl or a substituted alkyl. The substitution mayallow the conjugation to biomolecules. The molecules may be attached torhodamine dye core structural fragment through R₁₃ for further use.

Compounds of the invention may include the compound where R₁₆ and R₁₇are unsubstituted alkyl —(CH₂)_(n)H and —(CH₂)_(m)H where n is 1-3, R₁,R₄, R₅, R₆, R₈ and R₉ are all H, R₂ and R₃ are H or CH₃. Such compoundsare shown in formula (III) below:

where k, n, m are independently integers of from 1 to 3,q is an integer of from 1 to 6,R₂ and R₃ are independently H or CH₃,R₇ is OR₁₁ or NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or asubstituted alkyl, andR₁₃ is OR₁₄ or NR₁₄R₁₅ where R₁₄ and R₁₅ are independently H, alkyl or asubstituted alkyl; aryl or a substituted aryl.

Compounds of the invention may include the compound where R₁₆ and R₁₇are H, R₁ is linked to R₂ or R₈ via a chain of CH₂ groups to form aring, and R₄ is linked to R₃ or R₉ via a chain of CH₂ groups to form aring.

Compounds of the invention may include the compound where R₁₆ and R₁₇are H, R₁ is linked to R₂ via a chain of CH₂ groups to form a 6 memberedring, and R₄ is linked to R₃ via a chain of CH₂ groups to form a 6membered ring. Such compounds are shown in formula (IV) below:

where k, n, m are independently integers of from 1 to 3, where q is aninteger of from 1 to 6,R₇ is OR₁₁ or NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or asubstituted alkyl, andR₁₃ is OR₁₄ or NR₁₄R₁₅ where R₁₄ and R₁₅ are independently H, alkyl or asubstituted alkyl; aryl or a substituted aryl.

Dyes of the invention may include the compounds where any of R₁, R₄, R₁₆or R₁₇ are H or alkyl. The alkyl may be substituted with SO₃ ⁻ Compoundsof the invention may include the compound where R₁ and R₄ are H and R₁₆and/or R₁₇ is SO₃ ⁻.

The R₁₃CO—(CH₂)_(q)— group is joined to rhodamine dye core structuralfragment via a heteroatom, for example an oxygen atom. This heteroatom,may be attached to any of carbon atoms of the phenyl ring of therhodamine dye core structural fragment. The compounds may be preparedand used as mixtures of positional isomers where the heteroatom oroxygen atom is present at different positions of the benzene ring, orthe compounds may be prepared and used as single isomers. Optionally thebenzene ring of such new rhodamine dyes may contain additionalsubstituents for further fine tuning of their spectral parameters.

Compounds of the invention include compounds according to formula (Va)and (Vb), and mixtures thereof:

wherein M^(+/−) is a common counter ion,k is an integer of from 0 to 6,q is an integer of from 1 to 6,R₁ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,R₂ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group, or R₂ together with R₁ or R₅ is acarbon or heterosubstituted chain forming a ring,R₃ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group or R₃ together with R₄ or R₆ is acarbon or heterosubstituted chain forming a ring,R₄ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,R₅ and R₆ are H, alkyl or substituted alkyl group, halogen, hydroxy- oralkoxy group,R₈ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₁ is a carbon or heterosubstituted carbon chainforming a ring,R₉ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₄ is a carbon or heterosubstituted carbon chainforming a ring,R₇ is OR₁₁ or NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or asubstituted alkyl,R₁₃ is OR₁₄ or NR₁₄R₁₅ where R₁₄ and R₁₅ are independently H, alkyl or asubstituted alkyl; aryl or a substituted aryl, andR₁₆ and R₁₇ are independently H or an alkyl, aryl or substituted alkylor substituted aryl group.

The compounds of the invention may be attached to biomolecules. Thecompounds of the invention may be attached to oligonucleotides. Thecompounds of the invention may be attached to nucleotides. The compoundsmay be attached to oligonucleotides or nucleotides via the nucleotidebase.

The attachment to the biomolecules may be via one of COR₇ and/orR₁₃CO(CH2)q-O— group or via both of these. The R₇ and/or R₁₃ group maybe an alkyl-, aryl-, or heteryl-oxy groups, primary or secondary aminewith a substituted group R₁₅, which may be used for attachment. Inparticular one of COR₇ and/or R₁₃CO(CH2)q-O— group may be activatedester residue most suitable for further amide/peptide bond formation.

For example, R₇ and/or R₁₃ may be:

Examples of compounds of the invention include:

where q is an integer of from 1 to 6,R₇ is OR₁₁ or NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or asubstituted alkyl, andR₁₃ is OR₁₄ or NR₁₄R₁₅ where R₁₄ and R₁₅ are independently H, alkyl or asubstituted alkyl; aryl or a substituted aryl.

Further examples include

An aspect of the invention is a nucleotide or oligonucleotide labelledwith a fluorescent compound as described herein. The labelled nucleotideor oligonucleotide may have the label attached via substituted alkylgroup R₁₅. The labelled nucleotide or oligonucleotide may have the labelattached to the C5 position of a pyrimidine base or the C7 position of a7-deaza purine base through a linker moiety.

The labelled nucleotide or oligonucleotide may also have a 3′ OHblocking group covalently attached to the ribose or deoxyribose sugar ofthe nucleotide.

Disclosed herein are kits comprising two or more nucleotides wherein atleast one nucleotide is a nucleotide labelled with a compound of thepresent invention. The kit may comprise two or more labellednucleotides. The nucleotides may be labelled with two or morefluorescent labels. Two or more of the labels may be excited using asingle excitation source, which may be a laser.

The kit may contain four labelled nucleotides, where the first of fournucleotides is labelled with a compound as disclosed herein, and thesecond, third, and fourth nucleotides are each labelled with a differentcompound, wherein each compound has a distinct absorbance maximum andeach of the compounds is distinguishable from the other three compounds.The kit may be such that two or more of the compounds have a distinctabsorbance maximum above 600 nm.

The compounds of the invention, nucleotides or kits may be used insequencing, expression analysis, hybridisation analysis, geneticanalysis, RNA analysis or protein binding assays. The use may be on anautomated sequencing instrument. The sequencing instrument may containtwo lasers operating at different wavelengths.

Disclosed herein is new compounds of formula (Xa,b) as startingmaterials for synthesising of dyes formula (I) in according with theinvention.

where q is 1-6.

Disclosed herein is a method of synthesising compounds of the invention.A compound of formula (Xa,b) may be used in a condensation reaction witha substituted or unsubstituted 3-amino phenol derivative or heterocyclicderivative which contains this structural fragment. Dyes of formula (I)have been synthesized by a condensation of the phthalic acid derivatives(Xa,b) with 3-amino-phenol derivatives, preferably at high temperature,with or without Lewis acid catalysts. Reaction could be fulfilled alsoby using phosphoric or poly-phosphoric acids as solvent and/or acatalyst. Condensation reaction may be accomplished with or withoutcatalysts in ionic liquids or high-boiling point polar organic solventlike DMF, DMA, sulfolane or 1,2-dichlorobenzene.

As used herein, the term “alkyl” refers to C1-C20 hydrocarbon and mayinclude C3-C10 non-aromatic carbocyclic rings. In particular embodimentsthe alkyl groups are C1-C6 alkyl which refers to saturated, straight- orbranched-chain hydrocarbon radicals containing between one and sixcarbon atoms, respectively. Alkyl groups may include one or moreunsaturated groups, and thus include alkenyl and alkynyl.

The term “halogen” as used herein refers to fluoro-(hereafter designatedas F), chloro-(hereafter designated as Cl), bromo-(hereafter designatedas Br) or iodo-(hereafter designated as I), and usually relates tosubstitution for a hydrogen atom in an organic compound, thissubstitution is optionally a full substitution for the hydrogen.

The term “substituted alkyl”, refers to alkyl, alkenyl or alkynyl groupsas defined above where they may optionally be further substituted with,but not limited to, halo, cyano, SO₃ ⁻, SRa, ORa, NRbRc, oxo, CONRbRc,COOH and COORb. Ra, Rb and Rc may be each independently selected from H,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl and substituted aryl. Further, saidsubstituted alkyl, substituted alkenyl and substituted alkynyl mayoptionally be interrupted by at least one hetero atom or group selectedfrom O, NRb, S(O)_(t) where t is 0 to 2, and the like. Substituted alkylalso covers group such as benzyl where the alkyl groups is comprises afurther aryl or substituted aryl moiety.

Dyes according to the present invention may be synthesised from avariety of different starting materials, including N- and/orC-substituted derivatives of 3-aminophenol, 5-hydroxy- and/or7-hydroxy-1,2,3,4-tetrahydroquinoline derivatives and other similararomatic or heterocyclic starting materials which contain m-aminophenolstructural fragment. Condensation of these compounds with additionallysubstituted or unsubstituted phthalic acid derivatives formula Xa,bgives the dyes as described. The condensation reaction is typicallycarried out at high temperature with or without suitable solvent, and isassisted by the use of microwave irradiation. The use of ionic liquidsas solvent in said condensation reactions is especially advantageous.The reaction can be catalysed by Lewis acids.

Dyes according to the invention may be synthesised also by conventionalalkylation methods starting from hydroxy-substituted rhodamine dyesformula (XI)

M^(+/−) is a common counter ion,k is an integer of from 0 to 6,R₁ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,R₂ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group, or R₂ together with R₁ or R₅ is acarbon or heterosubstituted chain forming a ring,R₃ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group or R₃ together with R₄ or R₆ is acarbon or heterosubstituted chain forming a ring,R₄ is H or an alkyl, aryl or substituted alkyl or substituted arylgroup,R₅ and R₆ are H, alkyl or substituted alkyl group, halogen, hydroxy- oralkoxy group,R₈ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₁ is a carbon or heterosubstituted carbon chainforming a ring,R₉ is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₄ is a carbon or heterosubstituted carbon chainforming a ring,R₇ is OR₁₁ or NR₁₁R₁₂ where R₁₁ and R₁₂ are independently H, alkyl or asubstituted alkyl, andR₁₆ and R₁₇ are independently H or an alkyl, aryl or substituted alkylor substituted aryl group.

Dyes formula (XI) can be prepared by condensation of 3-aminophenolderivatives with hydroxyl-phthalic acid derivatives as described below.

Preparation of N-alkyl-5-hydroxy-1,2,3,4-tetrahydroquinoline orN-sulfonatoalkyl-7-hydroxy-1,2,3,4-tetrahydroquinoline can be carriedout using chemical or catalytic hydrogenation, for example using RaneyNickel as a catalyst. The addition of an organic or inorganic base, forexample triethylamine, greatly enhances the rate of reaction.

Mono N-alkylation of 3-aminophenols with alkylsultones can be carriedout using one equivalent or more of the aminophenol. Di-alkylation of3-amino phenol can be achieved using more equivalents of thealkylsultone. Both of the resultant phenolic derivatives can becondensed with phthalic anhydride to make fluorophores as described.

According to an aspect of the invention there are provided dye compoundssuitable for attachment to substrate moieties, particularly comprisinglinker groups to enable attachment to substrate moieties. Substratemoieties can be virtually any molecule or substance to which the dyes ofthe invention can be conjugated and, by way of non-limiting example, mayinclude nucleosides, nucleotides, polynucleotides, carbohydrates,ligands, particles, solid surfaces, organic and inorganic polymers andcombinations or assemblages thereof, such as chromosomes, nuclei, livingcells and the like. The dyes can be conjugated by an optional linker bya variety of means including hydrophobic attraction, ionic attractionand covalent attachment. Particularly the dyes are conjugated to thesubstrate by covalent attachment. More particularly the covalentattachment is by means of a linker group.

The dyes according to the invention may include a reactive linker groupat one of the substituent positions for covalent attachment of the dyeto another molecule. Reactive linking groups are moieties capable offorming a covalent bond. In a particular embodiment the linker may be acleavable linker. Use of the term “cleavable linker” is not meant toimply that the whole linker is required to be removed. The cleavage sitecan be located at a position on the linker that ensures that part of thelinker remains attached to the dye and/or substrate moiety aftercleavage. Cleavable linkers may be, by way of non-limiting example,electrophilically cleavable linkers, nucleophilically cleavable linkers,photocleavable linkers, cleavable under reductive conditions (forexample disulfide or azide containing linkers), oxidative conditions,cleavable via use of safety-catch linkers and cleavable by eliminationmechanisms. The use of a cleavable linker to attach the dye compound toa substrate moiety ensures that the label can, if required, be removedafter detection, avoiding any interfering signal in downstream steps.

Particular linker groups may be found in pending patent applicationnumber WO2004/018493 (herein incorporated by reference) wherein thepresent inventors have found that certain linkers which connect thebases of nucleotides to labels such as, for example, dyes according tothe present invention, may be cleaved using water-soluble phosphines orwater-soluble transition metal catalysts formed from a transition metaland at least partially water-soluble ligands. In aqueous solution thelatter form at least partially water-soluble transition metal complexes.

Particular linkers may be found in Applicants pending Internationalapplication WO2004/018493 (herein incorporated by reference) and maycomprise moieties of the formula:

(wherein X is selected from the group comprising O, S, NH and NQ whereinQ is a C1-10 substituted or unsubstituted alkyl group, Y is selectedfrom the group comprising O, S, NH and N(allyl), T is hydrogen or aC1-10 substituted or unsubstituted alkyl group and * indicates where themoiety is connected to the remainder of the nucleotide or nucleoside).

Still yet more particularly the inventors have determined in pending GBpatent application number 0517097.2, published as WO07020457, (hereinincorporated by reference) that by altering, and in particularincreasing, the length of the linker between a fluorescent dye(fluorophore) and the guanine base, by introducing a polyethylene glycolspacer group, it is possible to increase the fluorescence intensitycompared to the same fluorophore attached to the guanine base throughother linkages known in the art. The design of the linkers, andespecially their increased length, also allows improvements in thebrightness of fluorophores attached to the guanine bases of guanosinenucleotides when incorporated into polynucleotides such as DNA. Thus,when the dye is for use in any method of analysis which requiresdetection of a fluorescent dye label attached to a guanine-containingnucleotide, it is advantageous if the linker comprises a spacer group offormula ((CH₂)₂O)_(n)— wherein n is an integer between 2 and 50, asdescribed in applicants pending application number GB0517097.2(WO07020457).

The present invention further provides conjugates of nucleosides andnucleotides labelled with dyes according to the invention (modifiednucleotides). Labelled nucleosides and nucleotides are useful forlabelling polynucleotides formed by enzymatic synthesis, such as, by wayof non-limiting example, in PCR amplification, isothermal amplificationor solid phase amplification, polynucleotide sequencing including solidphase sequencing, nick translation reactions and the like.

Nucleosides and nucleotides may be labelled at sites on the sugar ornucleobase. As known in the art, a “nucleotide” consists of anitrogenous base, a sugar, and one or more phosphate groups. In RNA thesugar is ribose and in DNA is a deoxyribose, i.e. a sugar lacking ahydroxyl group that is present in ribose. The nitrogenous base is aderivative of purine or pyrimidine. The purines are adenine (A) andguanine (G), and the pyrimidines are cytosine (C) and thymine (T) or inthe context of RNA, uracil (U). The C-1 atom of deoxyribose is bonded toN-1 of a pyrimidine or N-9 of a purine. A nucleotide is also a phosphateester of a nucleoside, with esterification occurring on the hydroxylgroup attached to the C-3 or C-5 of the sugar. Nucleotides are usuallymono, di- or triphosphates.

A “nucleoside” is structurally similar to a nucleotide but is missingthe phosphate moieties. An example of a nucleoside analog would be onein which the label is linked to the base and there is no phosphate groupattached to the sugar molecule.

Although the base is usually referred to as a purine or pyrimidine, theskilled person will appreciate that derivatives and analogues areavailable which do not alter the capability of the nucleotide ornucleoside to undergo Watson-Crick base pairing. “Derivative” or“analogue” means a compound or molecule whose core structure is the sameas, or closely resembles that of a parent compound but which has achemical or physical modification, such as, for example, a different oradditional side group, which allows the derivative nucleotide ornucleoside to be linked to another molecule. For example, the base maybe a deazapurine. The derivatives should be capable of undergoingWatson-Crick pairing. “Derivative” and “analogue” also mean a syntheticnucleotide or nucleoside derivative having modified base moieties and/ormodified sugar moieties. Such derivatives and analogues are discussedin, for example, Scheit, Nucleotide analogs (John Wiley & Son, 1980) andUhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analoguescan also comprise modified phosphodiester linkages includingphosphorothioate, phosphorodithioate, alkyl-phosphonate,phosphoranilidate, phosphoramidate linkages and the like.

The dye may be attached to any position on the nucleotide base, througha linker, provided that Watson-Crick base pairing can still be carriedout. Particular nucleobase labelling sites include the C5 position of apyrimidine base or the C7 position of a 7-deaza purine base. Asdescribed above a linker group may be used to covalently attach a dye tothe nucleoside or nucleotide.

In particular embodiments the labelled nucleoside or nucleotide may beenzymatically incorporable and enzymatically extendable. Accordingly alinker moiety may be of sufficient length to connect the nucleotide tothe compound such that the compound does not significantly interferewith the overall binding and recognition of the nucleotide by a nucleicacid replication enzyme. Thus, the linker can also comprise a spacerunit. The spacer distances, for example, the nucleotide base from acleavage site or label.

Nucleosides or nucleotides labelled with dyes of the invention may havethe formula:

Where Dye is a dye compound according to the present invention, B is anucleobase, such as, for example uracil, thymine, cytosine, adenine,guanine and the like and L is an optional linker group which may or maynot be present. R′ can be H, monophosphate, diphosphate, triphosphate,thiophosphate, a phosphate ester analog, —O— attached to a reactivephosphorous containing group or —O— protected by a blocking group. R″can be H, OH, a phosphoramidite or a 3′OH blocking group and R′″ is H orOH.

Where R″ is phosphoramidite, R′ is an acid-cleavable hydroxyl protectinggroup which allows subsequent monomer coupling under automated synthesisconditions.

In a particular embodiment the blocking group is separate andindependent of the dye compound, i.e. not attached to it. In analternative embodiment the dye may comprise all or part of the 3′OHblocking group. Thus R″ can be a 3′OH blocking group which may or maynot comprise the dye compound.

In still yet another alternative embodiment there is no blocking groupon the 3′ carbon of the pentose sugar and the dye (or dye and linkerconstruct) attached to the base, for example, can be of a size orstructure sufficient to act as a block to the incorporation of a furthernucleotide from a point other than the 3′ site. Thus the block can bedue to steric hindrance or can be due to a combination of size, chargeand structure.

The use of a blocking group allows polymerisation to be controlled, suchas by stopping extension when a modified nucleotide is incorporated. Ifthe blocking effect is reversible, for example by way of non-limitingexample by changing chemical conditions or by removal of a chemicalblock, extension can be stopped at certain points and then allowed tocontinue.

In another particular embodiment a 3′OH blocking group will comprisemoieties disclosed in WO2004/018497 (herein incorporated by reference).For example the blocking group may be azidomethyl (CH₂N₃) or allyl.

In a particular embodiment the linker and blocking group are bothpresent and are separate moieties which are both cleavable undersubstantially similar conditions. Thus deprotection and deblockingprocesses may be more efficient since only a single treatment will berequired to remove both the dye compound and the block.

The invention also encompasses polynucleotides incorporating dyecompounds according to the present invention. Such polynucleotides maybe DNA or RNA comprised respectively of deoxyribonucleotides orribonucleotides joined in phosphodiester linkage. Polynucleotidesaccording to the invention may comprise naturally occurring nucleotides,non-naturally occurring (or modified) nucleotides other than themodified nucleotides of the invention or any combination thereof,provided that at least one modified nucleotide, i.e. labelled with a dyecompound, according to the invention is present. Polynucleotidesaccording to the invention may also include non-natural backbonelinkages and/or non-nucleotide chemical modifications. Chimericstructures comprised of mixtures of ribonucleotides anddeoxyribonucleotides comprising at least one modified nucleotideaccording to the invention are also contemplated.

Modified nucleotides (or nucleosides) comprising a dye compoundaccording to the invention may be used in any method of analysis whichrequires detection of a fluorescent label attached to a nucleotide ornucleoside, whether on its own or incorporated into or associated with alarger molecular structure or conjugate. In this context the term“incorporated into a polynucleotide” requires that the 5′ phosphate isjoined in phosphodiester linkage to the 3′ hydroxyl group of a second(modified or unmodified) nucleotide, which may itself form part of alonger polynucleotide chain. The 3′ end of the modified nucleotide ofthe invention may or may not be joined in phosphodiester linkage to the5′phosphate of a further (modified or unmodified) nucleotide. Thus, inone non-limiting embodiment the invention provides a method of detectinga modified nucleotide incorporated into a polynucleotide whichcomprises: (a) incorporating at least one modified nucleotide accordingto the third aspect of the invention into a polynucleotide and (b)detecting the modified nucleotide(s) incorporated into thepolynucleotide by detecting the fluorescent signal from the dye compoundattached to said modified nucleotide(s).

This method requires two essential steps: a synthetic step (a) in whichone or more modified nucleotides according to the invention areincorporated into a polynucleotide and a detection step (b) in which oneor more modified nucleotide(s) incorporated into the polynucleotide aredetected by detecting or quantitatively measuring their fluorescence.

In one embodiment of the invention the at least one modified nucleotideis incorporated into a polynucleotide in the synthetic step by theaction of a polymerase enzyme. However, other methods of joiningmodified nucleotides to polynucleotides, such as for example chemicaloligonucleotide synthesis or ligation of labelled oligonucleotides tounlabelled oligonucleotides, are not excluded. Therefore, in thespecific context of this method of the invention, the term“incorporating” a nucleotide into a polynucleotide encompassespolynucleotide synthesis by chemical methods as well as enzymaticmethods.

In a specific embodiment the synthetic step may comprise incubating atemplate polynucleotide strand with a reaction mixture comprisingfluorescently labelled modified nucleotides of the invention and apolymerase under conditions which permit formation of a phosphodiesterlinkage between a free 3′ hydroxyl group on a polynucleotide strandannealed to said template polynucleotide strand and a 5′ phosphate groupon said modified nucleotide. This embodiment comprises a synthetic stepin which formation of a polynucleotide strand is directed bycomplementary base-pairing of nucleotides to a template strand.

In all embodiments of the method, the detection step may be carried outwhilst the polynucleotide strand into which the modified nucleotides areincorporated is annealed to a template strand, or after a denaturationstep in which the two strands are separated. Further steps, for examplechemical or enzymatic reaction steps or purification steps, may beincluded between the synthetic step and the detection step. Inparticular, the target strand incorporating the modified nucleotide(s)may be isolated or purified and then processed further or used in asubsequent analysis. By way of example, target polynucleotides labelledwith modified nucleotide(s) according to the invention in a syntheticstep may be subsequently used as labelled probes or primers. In otherembodiments the product of the synthetic step (a) may be subject tofurther reaction steps and, if desired, the product of these subsequentsteps purified or isolated.

Suitable conditions for the synthetic step will be well known to thosefamiliar with standard molecular biology techniques. In one embodimentthe synthetic step may be analogous to a standard primer extensionreaction using nucleotide precursors, including modified nucleotidesaccording to the invention, to form an extended target strandcomplementary to the template strand in the presence of a suitablepolymerase enzyme. In other embodiments the synthetic step may itselfform part of an amplification reaction producing a labelled doublestranded amplification product comprised of annealed complementarystrands derived from copying of the target and template polynucleotidestrands. Other exemplary “synthetic” steps include nick translation,strand displacement polymerisation, random primed DNA labelling etc. Thepolymerase enzyme used in the synthetic step must be capable ofcatalysing the incorporation of modified nucleotides according to theinvention. Otherwise, the precise nature of the polymerase is notparticularly limited but may depend upon the conditions of the syntheticreaction. By way of example, if the synthetic reaction is carried outusing thermocycling then a thermostable polymerase is required, whereasthis may not be essential for standard primer extension reactions.Suitable thermostable polymerases which are capable of incorporating themodified nucleotides according to the invention include those describedin WO 2005/024010 or WO06120433. In synthetic reactions which arecarried out at lower temperatures such as 37.degree. C., polymeraseenzymes need not necessarily be thermostable polymerases, therefore thechoice of polymerase will depend on a number of factors such as reactiontemperature, pH, strand-displacing activity and the like.

In specific non-limiting embodiments the invention encompasses use ofthe modified nucleotides or nucleosides labelled with dyes according tothe invention in a method of nucleic acid sequencing, re-sequencing,whole genome sequencing, single nucleotide polymorphism scoring, anyother application involving the detection of the modified nucleotide ornucleoside when incorporated into a polynucleotide, or any otherapplication requiring the use of polynucleotides labelled with themodified nucleotides comprising fluorescent dyes according to theinvention.

In a particular embodiment the invention provides use of modifiednucleotides comprising dye compounds according to the invention in apolynucleotide “sequencing-by-synthesis” reaction.Sequencing-by-synthesis generally involves sequential addition of one ormore nucleotides or oligonucleotides to a growing polynucleotide chainin the 5′ to 3′ direction using a polymerase or ligase in order to forman extended polynucleotide chain complementary to the template nucleicacid to be sequenced. The identity of the base present in one or more ofthe added nucleotide(s) is determined in a detection or “imaging” step.The identity of the added base may be determined after each nucleotideincorporation step. The sequence of the template may then be inferredusing conventional Watson-Crick base-pairing rules. The use of themodified nucleotides labelled with dyes according to the invention fordetermination of the identity of a single base may be useful, forexample, in the scoring of single nucleotide polymorphisms, and suchsingle base extension reactions are within the scope of this invention.

In an embodiment of the invention, the sequence of a templatepolynucleotide is determined by detecting the incorporation of one ormore nucleotides into a nascent strand complementary to the templatepolynucleotide to be sequenced through the detection of fluorescentlabel(s) attached to the incorporated nucleotide(s). Sequencing of thetemplate polynucleotide is primed with a suitable primer (or prepared asa hairpin construct which will contain the primer as part of thehairpin), and the nascent chain is extended in a stepwise manner byaddition of nucleotides to the 3′ end of the primer in apolymerase-catalysed reaction.

In particular embodiments each of the different nucleotide triphosphates(A, T, G and C) may be labelled with a unique fluorophore and alsocomprises a blocking group at the 3′ position to prevent uncontrolledpolymerisation. Alternatively one of the four nucleotides may beunlabelled (dark). The polymerase enzyme incorporates a nucleotide intothe nascent chain complementary to the template polynucleotide, and theblocking group prevents further incorporation of nucleotides. Anyunincorporated nucleotides are removed and the fluorescent signal fromeach incorporated nucleotide is “read” optically by suitable means, suchas a charge-coupled device using laser excitation and suitable emissionfilters. The 3′-blocking group and fluorescent dye compounds are thenremoved (deprotected), particularly by the same chemical or enzymaticmethod, to expose the nascent chain for further nucleotideincorporation. Typically the identity of the incorporated nucleotidewill be determined after each incorporation step but this is notstrictly essential. Similarly, U.S. Pat. No. 5,302,509 discloses amethod to sequence polynucleotides immobilised on a solid support. Themethod relies on the incorporation of fluorescently labelled, 3′-blockednucleotides A, G, C and T into a growing strand complementary to theimmobilised polynucleotide, in the presence of DNA polymerase. Thepolymerase incorporates a base complementary to the targetpolynucleotide, but is prevented from further addition by the3′-blocking group. The label of the incorporated nucleotide can then bedetermined and the blocking group removed by chemical cleavage to allowfurther polymerisation to occur. The nucleic acid template to besequenced in a sequencing-by-synthesis reaction may be anypolynucleotide that it is desired to sequence. The nucleic acid templatefor a sequencing reaction will typically comprise a double strandedregion having a free 3′ hydroxyl group which serves as a primer orinitiation point for the addition of further nucleotides in thesequencing reaction. The region of the template to be sequenced willoverhang this free 3′ hydroxyl group on the complementary strand. Theoverhanging region of the template to be sequenced may be singlestranded but can be double-stranded, provided that a “nick is present”on the strand complementary to the template strand to be sequenced toprovide a free 3′ OH group for initiation of the sequencing reaction. Insuch embodiments sequencing may proceed by strand displacement. Incertain embodiments a primer bearing the free 3′ hydroxyl group may beadded as a separate component (e.g. a short oligonucleotide) whichhybridises to a single-stranded region of the template to be sequenced.Alternatively, the primer and the template strand to be sequenced mayeach form part of a partially self-complementary nucleic acid strandcapable of forming an intra-molecular duplex, such as for example ahairpin loop structure. Hairpin polynucleotides and methods by whichthey may be attached to solid supports are disclosed in applicant'sco-pending International application publication nos. WO0157248 and WO2005/047301. Nucleotides are added successively to the free 3′ hydroxylgroup, resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. The nature of the base which has been added may bedetermined, particularly but not necessarily after each nucleotideaddition, thus providing sequence information for the nucleic acidtemplate. The term “incorporation” of a nucleotide into a nucleic acidstrand (or polynucleotide) in this context refers to joining of thenucleotide to the free 3′ hydroxyl group of the nucleic acid strand viaformation of a phosphodiester linkage with the 5′ phosphate group of thenucleotide.

The nucleic acid template to be sequenced may be DNA or RNA, or even ahybrid molecule comprised of deoxynucleotides and ribonucleotides. Thenucleic acid template may comprise naturally occurring and/ornon-naturally occurring nucleotides and natural or non-natural backbonelinkages, provided that these do not prevent copying of the template inthe sequencing reaction.

In certain embodiments the nucleic acid template to be sequenced may beattached to a solid support via any suitable linkage method known in theart, for example via covalent attachment. In certain embodimentstemplate polynucleotides may be attached directly to a solid support(e.g. a silica-based support). However, in other embodiments of theinvention the surface of the solid support may be modified in some wayso as to allow either direct covalent attachment of templatepolynucleotides, or to immobilise the template polynucleotides through ahydrogel or polyelectrolyte multilayer, which may itself benon-covalently attached to the solid support.

Arrays in which polynucleotides have been directly attached tosilica-based supports are those for example disclosed in WO00006770,wherein polynucleotides are immobilised on a glass support by reactionbetween a pendant epoxide group on the glass with an internal aminogroup on the polynucleotide. In addition, Applicants disclose in aco-pending International patent application publication numberWO2005/047301 arrays of polynucleotides attached to a solid support,e.g. for use in the preparation of SMAs, by reaction of a sulphur-basednucleophile with the solid support. A still further example ofsolid-supported template polynucleotides is where the templatepolynucleotides are attached to hydrogel supported upon silica-based orother solid supports. Silica-based supports are typically used tosupport hydrogels and hydrogel arrays as described in WO00/31148,WO01/01143, WO02/12566, WO03/014392, U.S. Pat. No. 6,465,178 andWO00/53812.

A particular surface to which template polynucleotides may beimmobilised is a polyacrylamide hydrogel. Polyacrylamide hydrogels aredescribed in the prior art, some of which is discussed above. However, aparticular hydrogel is described in WO2005/065814.

DNA template molecules can be attached to beads or microparticles forthe purposes of sequencing; for example as described in U.S. Pat. No.6,172,218. Further examples of the preparation of bead libraries whereeach bead contains different DNA sequences can be found in the prior art(Nature. 437, 376-380 (2005); Science. 309, 5741, 1728-1732 (2005)).Sequencing of arrays of such beads using nucleotides as described iswithin the scope of the invention.

The template(s) to be sequenced may form part of an “array” on a solidsupport, in which case the array may take any convenient form. Thus, themethod of the invention is applicable to all types of “high density”arrays, including single-molecule arrays, clustered arrays and beadarrays. Modified nucleotides labelled with dye compounds of theinvention may be used for sequencing templates on essentially any typeof array formed by immobilisation of nucleic acid molecules on a solidsupport, and more particularly any type of high-density array. However,the modified nucleotides labelled with dye compounds of the inventionare particularly advantageous in the context of sequencing of clusteredarrays.

In multi-polynucleotide or clustered arrays, distinct regions on thearray comprise multiple polynucleotide template molecules. The term“clustered array” refers to an array wherein distinct regions or siteson the array comprise multiple polynucleotide molecules that are notindividually resolvable by optical means. Depending on how the array isformed each site on the array may comprise multiple copies of oneindividual polynucleotide molecule or even multiple copies of a smallnumber of different polynucleotide molecules (e.g. multiple copies oftwo complementary nucleic acid strands). Multi-polynucleotide orclustered arrays of nucleic acid molecules may be produced usingtechniques generally known in the art. By way of example, WO 98/44151and WO00/18957 both describe methods of amplification of nucleic acidswherein both the template and amplification products remain immobilisedon a solid support in order to form arrays comprised of clusters or“colonies” of immobilised nucleic acid molecules. The nucleic acidmolecules present on the clustered arrays prepared according to thesemethods are suitable templates for sequencing using the modifiednucleotides labelled with dye compounds of the invention.

The modified nucleotides labelled with dye compounds of the inventionare also useful in sequencing of templates on single molecule arrays.The term “single molecule array” or “SMA” as used herein refers to apopulation of polynucleotide molecules, distributed (or arrayed) over asolid support, wherein the spacing of any individual polynucleotide fromall others of the population is such that it is possible to effectindividual resolution of the polynucleotides. The target nucleic acidmolecules immobilised onto the surface of the solid support should thusbe capable of being resolved by optical means. This means that, withinthe resolvable area of the particular imaging device used, there must beone or more distinct signals, each representing one polynucleotide.

This may be achieved wherein the spacing between adjacent polynucleotidemolecules on the array is at least 100 nm, more particularly at least250 nm, still more particularly at least 300 nm, even more particularlyat least 350 nm. Thus, each molecule is individually resolvable anddetectable as a single molecule fluorescent point, and fluorescence fromsaid single molecule fluorescent point also exhibits single stepphotobleaching.

The terms “individually resolved” and “individual resolution” are usedherein to specify that, when visualised, it is possible to distinguishone molecule on the array from its neighbouring molecules. Separationbetween individual molecules on the array will be determined, in part,by the particular technique used to resolve the individual molecules.The general features of single molecule arrays will be understood byreference to published applications WO00/06770 and WO 01/57248. Althoughone use of the modified nucleotides of the invention is insequencing-by-synthesis reactions, the utility of the modifiednucleotides is not limited to such methods. In fact, the nucleotides maybe used advantageously in any sequencing methodology which requiresdetection of fluorescent labels attached to nucleotides incorporatedinto a polynucleotide.

In particular, the modified nucleotides labelled with dye compounds ofthe invention may be used in automated fluorescent sequencing protocols,particularly fluorescent dye-terminator cycle sequencing based on thechain termination sequencing method of Sanger and co-workers. Suchmethods generally use enzymes and cycle sequencing to incorporatefluorescently labelled dideoxynucleotides in a primer extensionsequencing reaction. So called Sanger sequencing methods, and relatedprotocols (Sanger-type), rely upon randomised chain termination withlabelled dideoxynucleotides.

Thus, the invention also encompasses modified nucleotides labelled withdye compounds according to the invention which are dideoxynucleotideslacking hydroxyl groups at both of the 3′ and 2′ positions, suchmodified dideoxynucleotides being suitable for use in Sanger typesequencing methods and the like.

Modified nucleotides labelled with dye compounds of the presentinvention incorporating 3′ blocking groups, it will be recognized, mayalso be of utility in Sanger methods and related protocols since thesame effect achieved by using modified dideoxy nucleotides may beachieved by using modified nucleotides having 3′-OH blocking groups:both prevent incorporation of subsequent nucleotides. Where nucleotidesaccording to the present invention, and having a 3′ blocking group areto be used in Sanger-type sequencing methods it will be appreciated thatthe dye compounds or detectable labels attached to the nucleotides neednot be connected via cleavable linkers, since in each instance where alabelled nucleotide of the invention is incorporated; no nucleotidesneed to be subsequently incorporated and thus the label need not beremoved from the nucleotide.

The invention also provides kits including modified nucleosides and/ornucleotides labelled with dyes according to the invention. Such kitswill generally include at least one modified nucleotide or nucleosidelabelled with a dye according to the invention together with at leastone further component. The further component(s) may be further modifiedor unmodified nucleotides or nucleosides. For example, modifiednucleotides labelled with dyes according to the invention may besupplied in combination with unlabelled or native nucleotides, and/orwith fluorescently labelled nucleotides or any combination thereof.Accordingly the kits may comprise modified nucleotides labelled withdyes according to the invention and modified nucleotides labelled withother, for example, prior art dye compounds. Combinations of nucleotidesmay be provided as separate individual components or as nucleotidemixtures.

Where kits comprise a plurality, particularly two, more particularlyfour, modified nucleotides labelled with a dye compound, the differentnucleotides may be labelled with different dye compounds, or one may bedark, with no dye compounds. Where the different nucleotides arelabelled with different dye compounds it is a feature of the kits thatsaid dye compounds are spectrally distinguishable fluorescent dyes. Asused herein, the term “spectrally distinguishable fluorescent dyes”refers to fluorescent dyes that emit fluorescent energy at wavelengthsthat can be distinguished by fluorescent detection equipment (forexample, a commercial capillary based DNA sequencing platform) when twoor more such dyes are present in one sample. When two modifiednucleotides labelled with fluorescent dye compounds are supplied in kitform, it is a feature of the invention that the spectrallydistinguishable fluorescent dyes can be excited at the same wavelength,such as, for example by the same laser. When four modified nucleotideslabelled with fluorescent dye compounds are supplied in kit form, it isa feature of the invention that two of the spectrally distinguishablefluorescent dyes can both be excited at one wavelength and the other twospectrally distinguishable dyes can both be excited at anotherwavelength. Particular excitation wavelengths are 532 nm, 630 nm to 700nm, particularly 660 nm.

In one embodiment a kit comprises a modified nucleotide labelled with acompound of the invention and a second modified nucleotide labelled witha second dye wherein the dyes have a difference in absorbance maximum ofat least 10 nm, particularly 20 nm to 50 nm. More particularly the twodye compounds have Stokes shifts of between 15-40 nm where “Stokesshift” is the distance between the peak absorption and peak emissionwavelengths.

In a further embodiment said kit further comprises two other modifiednucleotides labelled with fluorescent dyes wherein said dyes are excitedby the same laser at 600 nm to 700 nm, particularly 630 nm to 700 nm,more particularly 660 nm. Wherein the dyes have a difference inabsorbance maximum of at least 10 nm, particularly 20 nm to 50 nm. Moreparticularly the two dye compounds have Stokes shifts of between 20-40nm. Still yet more particularly the two dye compounds have a differentabsorbance maximum above 600 nm, particularly above 640 nm. Particulardyes which are spectrally distinguishable from rhodamine dyes of theinvention and which meet the above criteria are polymethine analogues asdescribed in U.S. Pat. No. 5,268,486 (for example Cy5) or WO 0226891(Alexa 647; Molecular Probes A20106) or unsymmetrical polymethines asdisclosed in U.S. Pat. No. 6,924,372.

In an alternative embodiment, the kits of the invention may containnucleotides where the same base is labelled with two differentcompounds. A first nucleotide may be labelled with a compound of theinvention. A second nucleotide may be labelled with a spectrallydistinct compound, for example a ‘red’ dye absorbing at greater than 600nm. A third nucleotide may be labelled as a mixture of the compound ofthe invention and the spectrally distinct compound, and the fourthnucleotide may be ‘dark’ and contain no label. In simple terms thereforethe nucleotides 1-4 may be labelled ‘green’, ‘red’, ‘red/green’, anddark. To simplify the instrumentation further, four nucleotides can belabelled with a two dyes excited with a single laser, and thus thelabelling of nucleotides 1-4 may be ‘green 1’, ‘green 2’ ‘green 1/green2’, and dark.

Nucleotides may contain two dyes of the present invention. Dyes where R₁and R₄ are H absorb at a lower wavelength than where R₁ and R₄ arealkyl. A kit may contain two or more nucleotides labelled with dyes ofthe invention. A kit may contain a nucleotide labelled with a compoundof the invention where R₁ and R₄ are H, and a second nucleotide labelledwith a compound of the invention where R₁ and R₄ are alkyl. Kits maycontain a further nucleotide where a portion of the nucleotide islabelled with a compound of the invention where R₁ and R₄ are H, and asecond portion of the nucleotide labelled with a compound of theinvention where R₁ and R₄ are alkyl. Kits may further contain anunlabelled nucleotide.

In other embodiments the kits may include a polymerase enzyme capable ofcatalyzing incorporation of the modified nucleotides into apolynucleotide. Other components to be included in such kits may includebuffers and the like. The modified nucleotides labelled with dyesaccording to the invention, and other any nucleotide componentsincluding mixtures of different nucleotides, may be provided in the kitin a concentrated form to be diluted prior to use. In such embodiments asuitable dilution buffer may also be included.

It is noted that, as used in this specification and the appended claims,the singular forms “a”, “an” and “the” include plural referents unlessexpressly and unequivocally limited to one referent. It will be apparentto those skilled in the art that various modifications and variationscan be made to various embodiments described herein without departingfrom the spirit or scope of the present teachings. Thus, it is intendedthat the various embodiments described herein cover other modificationsand variations within the scope of the appended claims and theirequivalents.

Experimental Details Diethyl 4-hydroxyphthalate (2.1)

This compound was prepared from 4-hydroxy-phthalic acid 2.0 by anesterification with an excess of ethanol (absolute) in the presence ofconcentrated sulphuric acid.

Preparation:

To a 0.5 L round bottomed flask containing absolute ethanol (250 ml,˜200 g, ˜4.3 mol) 4-hydroxyphthalic acid 2.0 (15 g, 82.4 mmol) was addedin a few portions at room temperature with stirring. This was thenfollowed by an addition of 0.5 ml (˜0.92 g, 9.4 mmol) of concentratedsulphuric acid. This mixture was stirred at room temperature for half anhour, and then it was further refluxed for 24 hours with a condenser.

About a half of solvent volume was distilled off using a descendedcondenser and the remaining solvent was removed under a reducedpressure. The viscous oily residue was dissolved in 400 ml DCM. To thissolution, 1.5 g anhydrous potassium carbonate was added carefully insmall portions. This mixture was kept at room temperature with stirringfor about 2 hours, than filtered through ˜100 g of silica gels. Thesilica gels were further washed with DCM to elute more absorbedproducts. The combined organic fractions were dried with anhydrousmagnesium sulphate (˜5 g) overnight. Solids were filtered off, andfurther washed with DCM. Solvent was evaporated under a reducedpressure.

This ester 2.1 obtained as colourless oily product was used in the nextstep without further purification.

Yield: 22 g (˜99%)

¹H NMR: (400 MHz, DMSO-d₆) δ 10.59 (s, 1H), 7.68 (d, J=8.5 Hz, 1H),7.07-6.76 (m, 2H), 4.22 (dq, J=13.4, 7.1 Hz, 4H), 1.25 (td, J=7.1, 3.3Hz, 6H).

Diethyl 4-[(3-ethoxycarbonyl)propyloxy]phthalate (2.2)

This compound was prepared in ethanol from diethyl 4-hydroxyphthalate2.1 via beforehand formation of potassium3,4-bis(ethoxycarbonyl)phenolate and then by alkylation with ethyl4-bromobutyrate in the same reaction pot.

Preparation:

To a solution of the ester 2.1 (2 g, 8.4 mmol) in absolute ethanol (20ml), solid potassium tert-butoxide (1.1 g, 9.8 mmol, 1.15 eq) was addedslowly. The reaction mixture was kept at room temperature for an hour,and then ethyl 4-bromobutyrate (2 g, ˜1.5 ml) was added. The reactionwas kept at room temperature for an hour, and then was heated underreflux with a condenser overnight.

The reaction mixture was chilled (up to about 0-5° C.) with an ice-bathfor 3-4 hours. Inorganic precipitates were filtered off and furtherwashed with a cold absolute ethanol. The precipitates were discarded andthe combined filtrates were concentrated under reduced pressure.

The residue that contained some inorganic solids was diluted with amixture of DCM-Petroleum ether (7:3, 50 ml) and filtered through of ashort plug of Silica Gels (˜10 g). Silica Gels were further washed withDCM-MeOH (97:3, ˜150 ml) to elute more absorbed product (TLC was used tomonitor the completion of product elution; use more eluent solvent ifneeded). The combined organic fractions were dried with anhydrousmagnesium sulphate (˜1 g) overnight and evaporated under a reducedpressure. This ester 2.2 was used in the next step without furtherpurification. Yield: 2 g (67.6%)

¹H NMR: (400 MHz, Methanol-d₄) δ 7.87-7.77 (m, 1H), 7.18-7.05 (m, 2H),4.33 (dq, J=14.5, 7.1 Hz, 4H), 4.21-4.07 (m, 4H), 2.53 (t, J=7.3 Hz,2H), 2.17-2.04 (m, 2H), 1.36 (td, J=7.1, 3.5 Hz, 6H), 1.25 (t, J=7.2 Hz,3H).

4-(3-Carboxypropyloxy)phthalic acid (2.3)

This derivative of phthalic acid was prepared by a saponification of thetriethyl ester 2.2 from previous step by an aqueous solution of sodiumhydroxide.

Preparation:

To a round bottomed flask, equipped with a stirrer and a condenser, 100ml de-ionised water was poured and NaOH (5 g, 125 mmol) were then addedin small portions. After sodium hydroxide was dissolved, the triethylester 2.2 (10.5 g, ˜30 mmol) was added. The reaction mixture wasvigorously stirred at 110° C. (heating block) for 5 hours. After about 3hours, the oily starting material 2.2 was dissolved. The reactionsolution was filtered and the filtrate collected was transfer to around-bottomed flask. About 10-15 ml of liquid containing ethanol formedduring the reaction and water, was distilled off with a descentcondenser. The remaining solution was cooled by ice-bath (0-5° C.).

To this cold solution, concentrated hydrochloric acid

(37%, 11 ml, 135 mmol) was added slowly with stirring. The pH of thismixture was checked by an indicator to ensure an excess of mineral acid.The expected product as a white precipitate was formed soon after this.The mixture was kept at ˜0-5° C. for an hour. The precipitate wasfiltered, washed with a few ml of cold water and then dried overnight.

This product 2.3 was used in the next step without further purification.However, it could be purified by crystallization from water ifnecessary. Yield: 5.1 g, 64%

¹H-NMR:

(400 MHz, CF₃COO-d) δ 11.62 (d, J=1.5 Hz, 6H), 8.10 (dd, J=8.8, 1.5 Hz,1H), 7.36 (d, J=2.4 Hz, 1H), 7.23-7.14 (m, 1H), 4.24 (td, J=5.9, 1.6 Hz,2H), 2.76 (td, J=7.2, 1.6 Hz, 2H), 2.35-2.24 (m, 2H).

4-(3-Carboxypropyloxy)phthalic anhydride (2.4)

Preparation of the anhydride 2.4 can be accomplished by reaction of theacid 2.3 with dehydrating reagents like acetic anhydride, preheating ofthe acid 2.3 at high temperatures or just by keeping the acid 2.3 in adissector with an inorganic dehydrating reagent.

4-Acetyloxyphthalic anhydride (2.5)

This compound was prepared from 4-hydroxy-phthalic acid 2.0 bydehydratation with an excess acetic anhydride in according withprocedure published in literature (Synthesis 2008, p. 3415).

To a solution of 4-hydroxyphthalic acid 2 (5.39 g) in toluene (100 ml)acetic anhydride (25 ml) was added and mixture was refluxed for 2 h.Next day the crystallized product was filtered off. Yield 2.6 g (42.5%).

The Dye (XI-1) Synthesis:

To a round-bottomed flask, 4-acetyloxyphtalic acid (1.03 g),3-ethylamino-cresol (2.0 g) and potassium pyrosulphate (1.5 g) wereadded. The reagents were carefully mixed together with a spatula andheated at 150° C. (heating block) for 6 h. During this time more andmore intensive red colour was developed and reaction mixture solidified.Both TLC (acetonitrile-water, 20%) and HPLC (@ 280 nm) were used tocheck the reaction progress by monitoring the ratio of dyes and thestarting 3-ethylaminocresol. After about 5 hours this ratio remainedroughly constant. To the reaction mixture 10 ml water was added and pinkcoloured product filtered off. The precipitate was placed into around-bottomed flask, methanol (50 ml) added and mixture refluxed for 10min. About a ¾ of the solvent was removed in vacuum and this redcoloured solution was left overnight. Product as red crystals wasfiltered off. Yield 1 g (46%).

If needed isomers may be separated by chromatography.

The Dye (I) Synthesis:

Target compounds as a mixture of constitutional isomers were synthesizedby a condensation of the phthalic acid 2.3 with7-hydroxy-1,2,3,4-tetrahydroquinoline at high temperature. Reactionfulfilled by using anhydrous phosphoric acid as a catalyst and solventas well.

Preparation:

4-(3-carboxypropoxy)phthalic acid (0.268 g) and 0.5 g of1-butyl-3-methyl-imidazolium hydrosulphate (IL-HSO₄) was heated 1 h at125° C. for completion of anhydride (Xa-2.4) formation.7-Hydroxy-tetrahydro-dihydroquinolin (0.298 g) was added and thereaction mixture was stirred at 175° C. for 2 h.

Red coloured compound with strong fluorescent formed. TLC control inCH₃CN-water (4:1)confirmed dyes formation. Heating at 175° C. wascontinued for additional 3 h.

This crude material was dissolved in CH₃CN-water mixture (10%) andpurified by flash column chromatography on Silica-gel. Red colouredfractions were collected and solvents evaporated.

Dye (I-1): Yield 18 mg (16%).

Dye (I-2): Yield 9 mg (8%).

Target compounds as a mixture of constitutional isomers were synthesizedby a condensation of the phthalic acid 2.3 with3-ethylamino-4-methyl-phenol at high temperature. Reaction fulfilled byusing anhydrous phosphoric acid as a catalyst with and withouthigh-boiling point polar organic solvent like DMF, DMA, sulfolane or1,2-dichlorobenzene.

This reaction can be carried out using potassium disulphate as acatalyst. This reagent acts as a mild Lewis acid, which may stimulatethe formation and/or further reactions of the starting materials and/orintermediates formed. As a result, the reaction temperature was lowerand the amount of side products was reduced. The nature of a catalystand a solvent been used exert a significant effect not only on thereaction yield and purity of the products but also on theregio-selectivity of the isomeric dyes formation. In this way formationof one or another isomer as a prevalent one can be achieved.

Additionally some ionic liquids (IL) can improve the rhodamine dyeformation both on reaction yield and product purity. We also haveachieved definitive improvement with some IL on the synthesis of thedyes (I). Among the IL (1-ethyl-3-methyl-imidazolium quaternary saltsfamily and some tetra-alkyl ammonium salts) we tested, some efficientlypromote the formation of one or another isomer and furnish a cleanerproduct (fewer coloured by-products in the reaction mixture).

Dyes (I-3) and (I-4)

Chemical Name:

(I-3):3,6-Bis(ethylamino)-2,7-dimethyl-[2-carboxylato-5-(3-carboxypropyloxy)phenyl]xanthyliumbetaine

(I-4):3,6-Bis(ethylamino)-2,7-dimethyl-[2-carboxylato-4-(3-carboxypropyloxy)phenyl]xanthyliumbetaine

Preparation:

To a round-bottomed flask, 4-carboxypropyloxyphtalic acid (1.0 g, 3.73mmol), 3-ethylamino-cresol (2.0 g, 13.23 mmol), IL-CF3 (1.72 g, 6.61mmol) and potassium pyrosulphate (1.68 g, 6.61 mmol) were added. Thereagents were carefully mixed together with a spatula and heated at 150°C. (heating block) for 10 h. During this time more and more intensivered colour was developed and reaction mixture solidified.

Both TLC (acetonitrile-water, 20%) and HPLC (@ 280 nm) were used tocheck the reaction progress by monitoring the ratio of dyes and thestarting 3-ethylaminocresol. After 10 hours this ratio remained roughlyconstant.

Reaction mixture was cooled to room temperature, and the red solid wasdissolved in methanol (˜20 ml). The colourless insoluble inorganicmaterials were filtered off. Triethylamine (2 ml) was added to thefiltrate and the resulting solution was applied to Biotage C₁₈ samplet(40 g). The samplet was then dried in vacuum to remove most of solvents.

Product was isolated by flash chromatography on Biotage Isolera-4instrument on a 400 g C₁₈ column using mixture of 0.1M TEAB in water andacetonitrile as eluting solvents (gradient 17-27). Product collectionwavelength was set at 520 nm, and the control wavelength was set at 280nm to monitor separation from excess of the starting materials andcolourless side products formed.

Yellow-orange coloured fractions were collected. Solvents were removedby a rotary evaporator in vacuum. The remaining solid red residue wastriturated with petroleum ether (60-95° C., 25 ml) and product, 1-3 as ared powder was filtered off and dried on air.

Yield: 0.50 g (29%).

(I-3):

¹H-NMR:

(400 MHz, DMSO-d₆) δ 7.84 (d, J=8.5 Hz, 1H), 7.18 (dd, J=8.5, 2.2 Hz,1H), 6.58 (d, J=2.2 Hz, 1H), 6.30 (s, 2H), 6.25 (s, 2H), 5.26 (t, J=5.4Hz, 2H), 3.95 (t, J=6.4 Hz, 2H), 3.22-3.10 (m, 4H), 2.30 (d, J=7.3 Hz,2H), 1.91 (s, 8H), 1.21 (t, J=7.1 Hz, 6H).

(I-4):

Yield: 0.16 g (9%).

Dye (I-5) I-3-NHS

Preparation:

The dye (I-3) was dried under high vacuum overnight then 35 mg sample(68 μmol) taken and was placed into round-bottomed flask. Anhydrous DMF(3 mL) and Hunig's base (678 μmol, 116 μL) was added to the flask.Mixture (became nearly colourless due to formation of cyclic derivative(IC) and was stirred for about 20 min under nitrogen atmosphere. TSTU(80 μmol, 25 mg) was then added. A light pink colour developed. Thereaction mixture was stirred at room temperature for 1 h,

Progress was checked by TLC, (20% H₂O in CH₃CN). This lactone could beisolated by precipitation from solution, or just distillation of thesolvent in vacuum. The NHS ester (I-5) prepared in this way was used forcoupling without further purification and isolation from solution. Ifneeded the NHS ester could be isolated from solution by precipitation,for example, by precipitation with ethyl acetate, filtered off anddried.

Dye (I-6) I-3-PEG12

Preparation:

A solution of NH₂Peg12COOH (203 μmol, 126 mg) in water (300 μL) wasadded to the solution of (I-5) prepared as described above and thereaction mixture was stirred at room temperature overnight. Completionof the reaction been checked by TLC (20% H₂O in acetonitrile). Aftercoupling completion, 0.1M TEAB solution in water (4 mL) was added andthe mixture was stirred at ˜20° C. for 1 h to quench reactiveintermediates. The solvents were removed under vacuum. Residue wasdissolved in 10 ml 0.1M TEAB. This solution filtered through a syringefilter 0.2 nm pore size and was transferred into two 5 mL HPLC vials forHPLC purification. The product was purified by HPLC using C18 reversephase column with acetonitrile-0.1M TEAB. Fractions with absorption max526 nm were collected. Yield 47 μmol (70%).

Dye (I-7) I-4-NHS

Preparation:

The dye (I-4) was dried under high vacuum overnight then 28 mg sampletaken. Anhydrous DMF (3 mL) and Hunig base was added to the flask.Mixture became nearly colourless. Solution of cyclic form been stirredfor about 20 min under nitrogen atmosphere. TSTU (25 mg) was then added.Light pink colour developed. Reaction mixture was stirred at RT for 1 h.Progress was checked by TLC, (20% H₂O in MeCN). NHS-ester R_(f) ˜0.7.Activation was completed in 2 h.

The NHS ester (I-7) prepared in this way was used for coupling withoutfurther purification and isolation from solution. If needed the NHSester could be isolated from solution by precipitation with ethylacetate, filtered off and dried.

Dye (I-8) I-4-PEG12

Preparation:

A solution of NH₂Peg12COOH (100 mg) in water (300 μL) was added to thesolution of (I-7) prepared as described above and this reaction mixturewas stirred at RT overnight. Completion of the reaction (NHS esterconsumption) been checked by TLC (20% H₂O in acetonitrile, plate driedin vac). To work up 0.1 M TEAB in water (4 mL) was added and the mixturewas stirred at ˜20° C. for 1 h. The solvents were removed under vacuum.Residue was dissolved in 10 ml 0.1 M TEAB. This solution filteredthrough a syringe filter 0.2 nm pore size and was transferred into two 5mL HPLC vials for purification by HPLC. The product was purified by HPLCusing C18 reverse phase column with acetonitrile-0.1 M TEAB. Fractionswith absorption max 520 nm were collected. Yield 41 μmol (76%).

Dye (I-9) pppG-I-3-PEG12

Preparation:

Anhydrous DMA (7 mL) and Hunig's Base (0.082 mL) were added to the driedsample (52 mg) of (I-6). A solution of TSTU (17 mg) in 1 mL of dry DMAwas then added. System was flushed two times with nitrogen and thenreaction mixture was stirred at room temperature for 1 h, According TLC(20% H₂O in CH₃CN) activation completed.

Once the activation is started a solution of pppG-LN3 (3 mL of stock 3.5mM) vac down to dryness. After activation was completed this solutionwas added to pppG. The reaction was stirred at room temperature undernitrogen atmosphere for 3 h. TLC control in 20% H₂O in acetonitrile.Reaction mixture was cooled down to −4° C. with an ice-bath, 0.1 M TEAB(4 mL) was then added and the mixture was stirred at room temperaturefor 10 min.

Purification: The solution was applied to column with ˜20 g ofsuspension of DEAE sephadex resin in 0.05 M TEAB solution and washedwith TEAB from 0.1 M up to 0.4 M. Coloured fractions were combined,co-evaporated with water to remove more TEAB and vac down to dryness.

The residue was then re-dissolve in TEAB 0.1 M. This solution wasfiltered through a syringe filter 0.2 nm pore size into a corning flask,and then transferred into high recovery 5 mL HPLC vials for HPLCpurification. The solution can be stored in the freezer untilpurification. The product was purified by HPLC using C18 reverse phasecolumn with acetonitrile-0.1 M TEAB as eluents, Fractions withabsorption 525 nm were collected. Yield: 54%.

Dye (I-10) pppG-I-4-PEG12

Preparation:

The dye (I-8) was dried after second prep HPLC purification anddissolved in anhydrous DMA (3 mL) and then Hunig's Base (0.036 g) wasadded. A solution of TSTU (11 mg) in 1 mL of dry DMA was then added.Reaction mixture was stirred at room temperature for 3 h. According TLC(20% H₂O in CH₃CN) the activation was completed.

A solution of pppG-LN3 (1.6 mL of stock 35.5 mM) was vac down todryness. After activation of (I-8) was completed this solution was addedto pppG. The reaction was stirred at room temperature under nitrogenatmosphere for 2 h. TLC (20% H₂O in acetonitrile) control indicated thereaction progress. Reaction mixture was kept at −4° C. overnight, 0.1 MTEAB (4 mL) was then added and the mixture was stirred at roomtemperature for 10 min.

Ion exchange purification: this solution was applied to column with ˜20g of suspension of DEAE sephadex resin in 0.05 M TEAB solution andwashed with TEAB from 0.1 M up to 0.35 M.

Coloured fractions eluted at about 0.35 M TEAB were combined,co-evaporated with water to remove more TEAB and vac down to dryness.

Purification: similar to previous compound. Yield: 14 μmol (51%).

Dye (I-11) pppT-I-3

Preparation:

Anhydrous DMA (15 mL) and Hunig's Base (0.06 mL) were added to the driedsample of the dye (I-3) (60 mg). Colourless solution of lactone IC wasformed. A solution of TSTU, (0.50 g) in 5 mL of dry DMA was then addedto this. The red colour of activated ester (I-6) developed. The reactionmixture was stirred at room temperature for 1 h, According TLC (20% H₂Oin MeCN) activation completed. After activation was completed thissolution was added to the solution of pppT-LN3 (0.23 g) in water (10mL). The reaction mixture was stirred at room temperature under nitrogenatmosphere for 3 h. Coupling progress was checked by TLC (20% H₂O inacetonitrile). Reaction mixture was cooled down to ˜4° C. with anice-bath, then solution of 0.1 M TEAB (5 mL) in water was added and themixture was stirred at room temperature for 10 min. The reaction mixturewas applied to column with ˜50 g of DEAE sephadex resin suspension in0.05 M TEAB solution in water and washed with TEAB (concentrationgradient from 0.1 M up to 0.5 M). Coloured fractions were collected andevaporated then co-evaporated again with water to remove more TEAB andvac down to dryness. The residue was then re-dissolve in TEAB 0.1 M.This solution was filtered through a syringe filter 0.2 nm pore sizeinto a corning flask and stored in the freezer. The product was purifiedby HPLC using C18 reverse phase column with acetonitrile-0.1 M TEAB, thefraction with absorption at 520 nm being collected. Yield 57%.

Dye (I-12)pppT-I-4

Preparation:

Anhydrous DMA (5 mL) and Hunig's Base (0.02 mL) were added to the driedsample of the dye (I-4) (20 mg). Colourless solution of lactone IC wasformed. A solution of TSTU, (0.175 g) in 1 mL of dry DMA was then addedto this. Red colour of activated ester (I-6) developed. The reactionmixture was stirred at room temperature for 1 h, According TLC (20% H₂Oin MeCN) activation completed. After activation was completed thissolution was added to the solution of pppT-LN3 (77 mg) in water (3 mL).The reaction mixture was stirred at room temperature under nitrogenatmosphere for 3 h. Coupling progress was checked by TLC (20% H₂O inacetonitrile). Reaction mixture was cooled down to −4° C. with anice-bath, then solution of 0.1 M TEAB (2 mL) in water was added and themixture was stirred at room temperature for 10 min. The reaction mixturewas applied to column with ˜20 g of DEAE sephadex resin suspension in0.05 M TEAB solution in water and washed with TEAB (concentrationgradient from 0.1 M up to 0.5 M). Coloured fractions were collected andevaporated then co-evaporated again with water to remove more TEAB andvac down to dryness. The residue was then re-dissolve in TEAB 0.1M. Thissolution was filtered through a syringe filter 0.2 nm pore size into acorning flask and stored in the freezer. The product was purified byHPLC using C18 reverse phase column with acetonitrile-0.1 M TEAB, thefraction with absorption at 520 nm being collected. Yield 57%.

Dye (I-13) pppT-I-1

Preparation:

Anhydrous DMA (10 mL) and Hunig's Base (0.04 mL) were added to the driedsample of the dye (I-1) (40 mg). Colourless solution of lactone IC wasformed. A solution of TSTU, (0.35 g) in 5 mL of dry DMA was then addedto this. Red colour of activated ester developed. The reaction mixturewas stirred at room temperature for 1 h. According to TLC (20% H₂O inMeCN), activation was completed. After activation was completed thissolution was added to the solution of pppT-LN3 (0.18 g) in water (10mL). The reaction mixture was stirred at room temperature under nitrogenatmosphere for 3 h. Coupling progress was checked by TLC (20% H₂O inacetonitrile). The reaction mixture was cooled down to ˜4° C. with anice-bath, then a solution of 0.1 M TEAB (5 mL) in water was added andthe mixture was stirred at room temperature for 10 min. The reactionmixture was applied to column with ˜50 g of DEAE sephadex resinsuspension in 0.05 M TEAB solution in water and washed with TEAB(concentration gradient from 0.1 M up to 0.5 M). Coloured fractions werecollected and evaporated then co-evaporated again with water to removemore TEAB and vac down to dryness. The residue was then re-dissolved inTEAB 0.1 M. This solution was filtered through a syringe filter 0.2 nmpore size into a corning flask and stored in the freezer. The productwas purified by HPLC using C18 reverse phase column withacetonitrile-0.1 M TEAB, fraction with absorption at 535 nm collected.Yield 60%.

Dye (I-14) pppT-I-2

Preparation:

Anhydrous DMA (10 mL) and Hunig's Base (0.04 mL) were added to the driedsample of the dye (I-2) (40 mg). Colourless solution of lactone IC wasformed. A solution of TSTU, (0.35 g) in 5 mL of dry DMA was then addedto this. Red colour of activated ester developed. The reaction mixturewas stirred at room temperature for 1 h. According to TLC (20% H₂O inCH₃CN), activation was completed. After activation was completed thissolution was added to the solution of pppT-LN3 (0.18 g) in water (10mL). The reaction mixture was stirred at room temperature under nitrogenatmosphere for 3 h. Coupling reaction progress was monitored by TLC (20%H₂O in acetonitrile). The reaction mixture was cooled down to ˜4° C.with an ice-bath, then a solution of 0.1 M TEAB (5 mL) in water wasadded and the mixture was stirred at room temperature for 10 min. Thereaction mixture was applied to column with ˜50 g of DEAE sephadex resinsuspension in 0.05 M TEAB solution in water and washed with TEAB(concentration gradient from 0.1 M up to 0.5 M). The coloured fractionswere collected and evaporated then co-evaporated again with water toremove more TEAB and vac down to dryness. The residue was thenre-dissolved in TEAB 0.1 M. This solution was filtered through a syringefilter 0.2 nm pore size into a corning flask and stored in the freezer.The product was purified by HPLC using C18 reverse phase column withacetonitrile-0.1M TEAB, the fraction with absorption at 535 nm beingcollected. Yield 45%.

Characterisation of New Dyes Vs Known Dyes

Temperature Intensity

Normalized fluorescence intensities of 1.10⁻⁶ M solutions of dyes (I-1)and (I-3) were compared with commercially available dye Atto532 for thesame spectral region at different temperature. The intensity of the dyesat 20, 40 and 60° C. were measured. FIG. 1 shows the relative intensityof the dyes at each temperature. The commercial dye Atto532 shows agreater loss of fluorescence intensity at higher temperatures relativeto the I-1 and I-3 dyes. FIG. 1 demonstrates that the fluorescence ofthe new dyes in water based solutions is less variable with thetemperature.

Intensity when Conjugated to Nucleotides

Normalised Fluorescence spectra of 1.10⁻⁶ M solutions of dye-nucleobaseconjugates (I-13)-T and (I-11)-T were compared with structural analoguewhen pppT is conjugated with commercially available dye Atto532.

FIG. 2 demonstrates that fluorescence of the nucleobase conjugates basedon these new dyes in water based solutions higher than commerciallyavailable dyes when excited by 532 nm light. The 1-3 dye is brighterthan the atto 532 dye at the same nucleotide concentration. The I-1 dyeis red shifted compared to the atto 532 dye.

FIG. 3 demonstrates that fluorescence of new dyes in water basedsolutions is less dependable on temperature. Normalized fluorescenceintensities 1.10⁻⁶ M solutions of dyes when conjugated with nucleobase,T-(I-11) and T-(I-13) as compared with commercially available dyeAtto532 conjugated with the same T-nucleobase. Both the I-1 and 1-3 dyesshow a higher fluorescence intensity at elevated temperatures comparedto atto-532.

Sequencing Data

FIG. 4 demonstrates better distinguishing of fluorescence signals when anucleobase been labelled with the new dye in according with theinvention (I-3) (lane 6) as compared with standard fluorophore set whenthe same nucleobase been conjugated with commercially available dyeAtto532 (control 1). FIG. 4 shows a plot of red intensity vs greenintensity in an Illumina 4 colour sequencing run. The higher distancebetween the groups of dyes signals lowers the chances of a miss-call,and therefore increases the accuracy of sequencing. The increase inbrightness of the 1-3 dye compared to the commercial dye means thesequencing data is improved.

The invention claimed is:
 1. A compound of formula (I) and mesomersthereof:

Wherein M^(+/−) is a common counter ion, k is an integer of from 0 to 6,q is an integer of from 1 to 6, R₁ is H or an alkyl, aryl or substitutedalkyl or substituted aryl group, R₂ is H, alkyl or substituted alkylgroup, halogen, carboxy, carboxamide, hydroxy- or alkoxy group, or R₂together with R₁ or R₅ is a carbon or heterosubstituted chain forming aring, R₃ is H, alkyl or substituted alkyl group, halogen, carboxy,carboxamide, hydroxy- or alkoxy group or R₃ together with R₄ or R₆ is acarbon or heterosubstituted chain forming a ring, R₄ is H or an alkyl,aryl or substituted alkyl or substituted aryl group, R₅ and R₆ are H,alkyl or substituted alkyl group, halogen, hydroxy- or alkoxy group, R₈is H, halogen, hydroxy- or alkoxy group, alkyl or substituted alkylgroup or together with R₁ is a carbon or heterosubstituted carbon chainforming a ring, R₉ is H, halogen, hydroxy- or alkoxy group, alkyl orsubstituted alkyl group or together with R₄ is a carbon orheterosubstituted carbon chain forming a ring, R₇ is OR₁₁ or NR₁₁R₁₂where R₁₁ and R₁₂ are independently H, alkyl or a substituted alkyl, R₁₃is OR₁₄ or NR₁₄R₁₅ where R₁₄ and R₁₅ are independently H, alkyl or asubstituted alkyl; aryl or a substituted aryl, and R₁₆ and R₁₇ areindependently H or an alkyl, aryl or substituted alkyl or substitutedaryl group.
 2. The compound of claim 1 wherein R₁₆ is alkyl and R₁₇ isalkyl.
 3. The compound of claim 1 wherein R₁, R₄, R₅, R₆, R₈ and R₉ areall H, R₂ and R₃ are H or CH₃.
 4. The compound of claim 1 wherein R₁₆and R₁₇ are H.
 5. The compound of claim 1 wherein R₁ is linked to R₂ orR₈ via a chain of CH₂ groups to form a ring, and R₄ is linked to R₃ orR₉ via a chain of CH₂ groups to form a ring.
 6. The compound of claim 5wherein R₁ and R₂ form a six membered ring, R₃ and R₄ form a sixmembered ring, R₅, R₆, R₈ and R₉ are H.
 7. The compound of claim 1wherein one or more of R₁, R₄, R₁₆ and R₁₇ are alkyl groups substitutedwith an SO₃ ⁻ group.
 8. The compound of claim 1 wherein R₇ is OH.
 9. Thecompound of claim 1 wherein q is
 3. 10. The compound of claim 1 whereinR₁₃ is OH.
 11. The compound of claim 1 wherein R₁₃ is NR₁₄R₁₅ where R₁₄is H or alkyl and R₁₅ is alkyl or a substituted alkyl.
 12. The compoundof claim 11 wherein the compound is attached to a nucleotide oroligonucleotide via substituted alkyl group R₁₅.